Enzyme for treatment and prevention of bloat

ABSTRACT

Administration of an enzyme obtained from the fermentation of a specific Streptomyces griseus is effective in preventing and/or curing bloat in ruminants.

United States Patent Hahn et al. Aug. 5, 1975 ENZYME FOR TREATMENT AND[51] Int. Cl. C121) 13/10; A61K 19/00 PREVENTION OF BLOAT [58] Field OfSearch 195/62, 65, 66 R [75] Inventors: Peter A. Hahn, Grosse PointPark,

Mich.; Frank J. Hartdegen, References Cited Columbia; Marlin A.Espenshade, UNITED STATES PATENTS how of 3,683,069 4/1972 Hooreman424/94 [73] Assignee: W. R. Grace & Co., New York,

Primary Examiner-Lionel M. Shapiro [22] Filed: Oct. 31, 1974 Attorney,Agent, or FirmCharles L. Harness 21 AppL No.2 519,449 [57] ABSTRACTRelaed Applicatinn Data Administration of an enzyme obtained from thefer- Division of 56K 422,709, 1360- 7, 1973 mentation of a specificStreptomyces griseus is effec- US. Cl. 195/62; 195/66 R tive inpreventing and/or curing bloat in ruminants.

3 Claims, N0 Drawings ENZYME FOR TREATMENT AND PREVENTION OF BLOAT Thisis a division of application Ser. No. 422,709 filed Dec. 7, 1973 now US.Pat. No. 3,868,448.

This invention is directed to curing or preventing bloat, especiallyfeedlot bloat, in ruminants by the administration of an enzyme obtainedby fermentation of a Streptomyces isolated from soil. This microorganismis herein referred to as NRRL 5747. It has been placed on deposit at US.Department of Agriculture, Northern Regional Research Laboratory, 1815North University Street, Peoria, Illinois. 61604, and is available fromthat depository under the conditions stated in Notice, 886 0.6. 638.

Taxonomy of NRRL 5747 This particular species of Streptomyces griseusforms loosely filamentous colonies with an abundance of long, branchingaerial hyphae that segment into spiral chains of bead-like spores.Whole-cell hydrolysates of this strain contain the LL-form ofdiaminopimelic acid.

The physiological properties of NRRL 5747 are listed in the table below,in comparison with 123 strains of other varieties of Streptomycesgriseus.

Table l W: of 123 known strains of S. grixeus having Response ofProperty the property NRRL 5747 Decomposition of Adenine 98 Casein 100Hypoxanthinc 100 Tyrosine 100 Urea 97 Xanthinc 100 Growth at 50 40 33100 Survival at 50 for 8 hr 99 Nitrite from nitrate 73 Utilization ofCitrate 99 Lactate 90 Malatc 100 Mucate 0 Oxalate 0 Succinate 100Resistance to Lysozyme l Salicylatc 2 Oxidation of glucose 98Fermentation of glucose 0 Hydrolysis of Hippurate 5 Starch 100 Acid fromAdonitol 43 Arabinosc 63 Dulcitol 0 Erythritol l Galactose 99 Glucose100 lnositol Lactose 100 Maltose 100 Mannitol 97 Mannose 100 Melibiosc 0mMethyl-D-glucoside 98 Raffinosc 2 Rhamnose 3l Sorbitol 0 Trehalose 99Xylosc 100 The enzyme used in this invention is obtained by fermentationof the aforesaid NRRL 5747. The enzyme is isolated from the culture incrude or in purified form and then'is administered to the ruminant in acontrolled dosage. A suitable dosage is 200 to 4000 or more units peranimal. A preferred dosage is 1000 to 2000 units per animal. The dosagecan be repeated periodically (for example at 30 minute intervals) untilthe bloat is relieved. In most cases one dose is sufficient. A unit ofenzyme obtained by the process of this invention is stated herein as avalue obtained by the effect of the enzyme on gastric mucin. Moreprecisely stated, a unit of enzyme is selected as that amount of enzyme(whether crude or pure) which, when added to 1 ml. of water, and thatsolution added to 1 m1. of Prepared Solution of Gastric Mucin (ashereinafter defined), reduces the viscosity of the said PreparedSolution of Gastric Mucin by 50% in 2 hours at 39 C.

The invention is directed to the process of fermenting the statedspecies of Streptomyces; to the enzyme so prepared; and to the processof preventing or relieving bloat in ruminants by the use of the saidenzyme.

Bloat can occur from pasture feeding as well as in the feedlot. Inpasture feeding bloat may be typically encountered when the ruminant isfed on lush legume pastures. The chain of causation is not clearlyunderstood, but it is speculated that lush legumes contain considerableamount of pectins, plant proteins, and saponins, some or all of whichcontribute to a stable foam.

Feedlot bloat can occur anywhere; it is independent of geography orlocale.

There appear to be some types of bloat that are brought about by highactivity of a gas-forming bacteria. Treatment with antibiotics may bringthis type of foam under control, by striking directly at thefoamproducing bacteria. In such cases the antibiotic can be addeddirectly to the feed or can be administered intramuscularly. Penicillinis an example. This type of treatment works better with mild bloat. Itdoes not appear to control cases of moderate or severe bloat. The use ofour enzyme material will control bloat whether mild, moderate or severe.

Feedlot bloat apparently may occur from abrupt changes in diet tostarchy concentrate feeds. Also, some feeds, e.g., barley, seem topromote bloat.

The instant invention is particularly useful in control of feedlotbloat.

The instant invention involves administration of an enzyme-containingproduct to the ruminant. This enzyme is able to break down the rumenfoam, thereby destroying the foam and permitting release of gas bynormal eructation reflex.

Our enzyme is so effective that the problem of maintaining apredetermined concentration of several hours time is completely avoided.In other words the enzyme in most cases works immediately. In the moreobstinate cases, a second treatment may be required. When our enzymebrings the foam under control, either by the first treatment or by asecond treatment, the cure appears to be substantially permanent, andthe bloat does not reappear.

Cause of Bloat The ultimate causes of bloat in a ruminant have yet to befirmly established. The condition itself consists of development of foamwithin the rumen or paunch of the animal. The gas phase of this foam maybe largely carbon dioxide admixed with other fermentation gases, e.g.,methane. The liquid continuous phase of the foam is itself generally anaqueous solution or suspension of very complex organic material,typically proteins, pectins, etc. But for the presence of the liquidphase of the foam, the animal would be able to void the gas by simpleeructation reflex; however, when foam covers the cardia area (entry ofthe esophagus into the rumen), the eructation reflex is inhibited. Hencethe pressure continues to build up, and if the animal does not obtainrelief it will die. In a severe case of bloat gas pressure may be ashigh as 70 mm. of Hg. This pressure results in an increased absorptionof carbon dioxide into the blood and restricts the return of the venousblood through the vena cava vein back to the heart. Under theseconditions, if sufficiently long continued, death occurs by suffocation.

The reason that the eructation reflex is inhibited when the cardia iscovered with foam is that if the animal were to eructate under suchconditions, some of the liquid in the foam would pass into the lungs.This, of course, would be very dangerous.

If the foam can be brought under control, so that it recedes from thecardia area, the normal eructation reflex will then be activated, andthe excess gas pressure can be relieved by normal eructation. A numberof legume bloat control measures do in fact involve foam control. It hasbeen found for example that treatment of the animal with an anti-foamingagent may relieve legume bloat but not feedlot bloat.

Some of the anti-foaming agents may act in a more complex manner. Thealkyl aryl sulfonates apparently act as inhibitors for pectin methylesterase. The function and efficacy of some of the other agents is notso clear. Other materials reported for foam control include vegetableoils, lecithin, animal fats, whale oil, mineral oils, liquid paraffins,paraffin-wax emulsions, detergents, turpentine, diethyl ether,silicones, glycerol, plant and animal mucins, and the like. However,materials that operate in legume bloat apparently do not workeffectively for feedlot bloat, and some treatments may actually bedetrimental. For example 8% soy bean oil (which works to relieve legumebloat) actually increased bloating in a case of attempted treat ment offeedlot bloat. Although preventives for feedlot bloat have been proposed(certain quaternary ammonium compounds-cf. U.S. Pat. No. 3,686,416)prior to the instant invention there was no known effective cure forfeedlot bloat. While we do not wish to be bound to any specificmechanism, we believe that our treatment works because our enzyme ineffect destroys the mucin that forms the continuous liquid phase of thebloat-foam. This is an unobvious approach with an unexpectedconsequence.

Michel Hooreman, in US. Pat. No. 3,683,069 describes the effect ofcertain enzymes (e.g., from Streptomyces fradiae) on the reduction ofviscosity of bronchial, intestinal, and cervical mucus for the purposeof improving nutrition, etc. He concludes that the enzyme should bechosen so as to give an effect intermediate between the effect given bytrypsin snd chymotrypsin. He reports work done with a more active enzymeobtained from Bacillus subtilis and shows that the use of that enzymewas deleterious. (It retarded growth in rats.) On the other hand theenzyme of the instant invention is more active toward mucus than thosedescribed by Hooreman, being even more active than that Hooremanobtained from Bacillus subtilis. Yet, not only has the use of our enzymenot retarded growth in ruminants, it has been remarkably effective in abeneficial way, i.e., in curing feedlot bloat. Our NRRL 5747, being a Streptomyces griseus, is of course a different microorganism fromStreptomyces fradiae.

The following examples illustrate without limiting the invention.

EXAMPLE 1 Culture Used to Produce the Enzyme NRRL 5747, used for theproduction of the enzyme, is a Streptomycete soil isolate. The organismgrows well on potato dextrose agar (PDA) and on Czapek Dox Agar (CDA).The organism is preserved in lyophilized ampuls.

Fermentation Production Process A. lnoeulum Stage The spores from atwo-week old agar slant (CDA) are suspended'in 10 ml. of sterilizeddeionized water, which is used to inoculate 1 liter of broth in a4-liter aspirator bottle. The medium is composed of normal fermentationconstituents, as follows:

Table 2 87% Concentrate Feed" Cottonseed meal Corn oil meal Soybean OilMeal (Solvent Extracted) 44% Protein K HPO FeSO -7H O CaCO NaCl

Deionized Water, balance to make l()0% pH adjusted to 7.0 beforesterilization Antifoaming agent (silicone oil) l drop/l00 ml. 30 minutessterilization at l2lC (25().F.), 15 psi Vitamin Premix 4,500,000 unitsVitamin A per lb. premix l,000,0()() units Vitamin D 500 units Vitamin EThe inoculum bottle is incubated on a rotary shaker (250 RPM) at 25C for3 days, and then checked for sterility before being crosscd" ortransferred to production fermentor.

A above for constituents) isolation of the Enzyme The enzyme isrecovered by utilizing the fact that it is insoluble in slightly acidicacetone Thus, the following steps are undertaken. First, thefermentation broth is centrifuged to remove all insolublernaterialincluding cells. The pH of the supernatant solution is then adjusted to5.5 by the addition of glacial acetic acid. The enzyme is precipitatedby adding} liters of acetone per one liter of solution. Optimum yield'is obtained by using cold acetone and cooling the suspension to for 18hours. The acetone insoluble enzyme is separated by centrifugation. Theresultant enzyme is then washed with dry acetoneto remove residualwater. The enzyme can also be obtained by any standard biochemicaltechniques, such as ammonium sulfate precipitation, ion exchange or gelfiltration chromatography, lyophylization, etc. Finally the enzyme isdried in vacuo at C. to remove acetone. This final product can be placedin a standard gelatin bolus for use. It is stable at room temperature.One gram of product so prepared generally represents about 500 units. ai

The Bloat-Enzyme Assay A. Substrate: Prepare a 7.5% (w/w) solution ofgastric mucin, bacteriological in 0.2 Mphosphate buffer, pH 7.0, byheating the suspension to around 50 C. until it dissolves, then coolingto 25 C.

B. Enzyme: The test enzyme solution can. be either centrifugedfermentation broth (per-*Example 1) used directly, or a solution of thedry isolated enzyme; 2 to 3 mg/mlis a good level if-a single-level assayis being done.

C. Hydrolysis: Pipette 1.0 ml. of substrate into each of two 25 ml.Erlenmeyer flasks. Pipette 1.0 ml. test solution into one of the flasks(*test) and 1.0 ml. water into the other (control). Place flasks into a39 Cv water bath and shake for 2 hours.

D. Assay: Draw the sample into a 1.0 ml. constricted tip graduatedpipette to the 0 mark. Release vac uum and measure the time in secondsrequired for the solution to drain to the 0.8 ml. mark. Do for bothtest, control and also for water (blank). NOTE: If drain time forcontrol is less than 13 see, prepare fresh substrate and repeat assay.This method gives accurate and reproducible results; however, anystandard method of measuring viseosity, e.g., Ostwald viscositimeterwith a constant temperature bath, is suitable.

E. Calculation of Enzyme Units: First, calculate the percent decrease inviscosity of mucin caused by the enzyme:

71 Viscosity (Time of Control)(Time of Sample) X 100 Decrease (Time ofControl)-(Time of Blank) Second, calculate the enzyme units in testsample. One unit of activity is defined as the amount of enzyme whichwhen added to 1.0 ml water gives an assay of 50% using the above test.The enzyme units are most accurately determined by varying the enzymeconcentration in the test solution until a 50% assay result is obtained.However, in any assay where the percent decrease as determined above isbetween 10 and the units can be calculated as given below.

The viscosity of a mucin solution is not a linear function of the mucinconcentration; therefore, the percent viscosity decrease of a mucinsolution is not a linear function of the amount of enzyme present.Therefore, the enzyme units must be determined from the quadraticequation of thehyperbolic'function relating degree of hydrolysis toviscosity change. To simplify calculations, the units as a function ofpercent viscosity decrease are given in Table 1 below.

Results are expressed in units/m1 for spun fermentation broths orunits/mg for solutions prepared from dry enzyme.

Enzyme Characteristics A. Yield: Typical fermentations give 2 to 6 gm.dry isolated enzyme per liter of centrifuged broth. The activity rangeis between 0.3 and 1.0 units/mg.

B. In vitro Testing: The above assay procedure uses bacteriologicalmucin as a substrate since its low cost makes it suitable for routinework. For an in vitro test, the enzyme activity was measured using assubstrate a solution of bovine submaxillary mucin. With this substrate,the percent viscosity decrease was four times that achieved usingbacteriological mucin on a per mg basis.

C. Stability: The degree of enzyme denaturation in the dry form wasdetermined as a function of time and temperature (i.e., storagestability). Storage at 43 (109F) for 28 weeks or at 50 C. (122F) for 8weeks resulted in less than a 10% activity decrease.

Administration of Enzyme There are several ways of administering theenzyme to the ruminant:

a. A convenient means is by bolus. A bolus is prepared by compressing200-2000 units of dry enzyme with sufficient dry compatible carrier,e.g., lactose, to make a bolus pellet about three-fourths inch indiameter by 3 inches long. This bolus can then be forced down theruminants throat by a conventional bolus gun. The idea, of course, is toget the bolus past the tongue. After this the animal will swallow themedication by reflex.

b. Another means is to fill a gelatin capsule with the requisite amountof enzyme, plus a dry compatible filler, e.g., lactose, and then thecapsule is likewise forced down the animals throat by a bolusgunsCapsule dimensions may be typically three-fourths inch diameter by 3inches long.

c. Another means is to inject the requisite dosage by hypodermic syringedirect into the rumen. The material injected is a liquid, comprising200-2000 units of en-.

zyme in a compatible liquid carrier, e.g., mineral oil. In

a typical case, 4 grams of dry enzyme (2000 units) were suspended in 16ml. of mineral oil and used in a 14- gauge hypodermic syringe.

d. The dried enzyme can also be pelletized either alone or as part of apre-mix and added asa supplement to the feed. The dried enzyme as apowder can added directly to the feed.

e. The broth (i.e., the final fermentation liquor containing the enzyme,and the broth being either whole or the liquid resulting fromcentrifugation or filtration) can be added directly to the feed. In thisfashion neither drying nor extraction is required. While this means canbe used as a medication, it is not one of the preferred modes ofadministration because of the animals reluctance to eat while it isbloated. This mode, however, is recommended as the simplest forprophylactic purposes. Sufficient enzyme product is added direct to thedaily feed of the ruminant to providethe recommended 200-2000 units ofenzyme.

EXAMPLE 1 TREATMENT OF TWO STEERS Four-gram gelatin capsules ofbloat-enzyme were effective in treating feedlot bloat in these twoinstances. The two steers in question had developed feedlot bloat as aresult of maladjustment to a high level starch concentrate feed (87%sorghum and 13% roughage). The customary symptoms of bloat were presentin both steers, namely, a severely distended rumen, and they were bothoff feed. One of the steers was given a single capsule of the bloatenzyme product (2000 units), and responded to this treatment withinfifteen minutes. In this steer the condition was considered cured, sincethe animal resumed normal feeding and all symptoms of bloat disappeared.

The second bloated steer was first let down" with a hose, but thisanimal immediately bloated again. One

capsule was administered first. Apparently this was not sufficient sinceit had no apparent effect after fifteen minutes time. A second capsulewas then administered. The bloated condition was completely relievedwithin 10 minutes of the second administration.

There was no recurrence of bloat for either of these two steers.

EXAMPLE 2 EXPERIMENT WITH A SINGLE STEER This animal, while not achronic bloater, had previously bloated, and on that occasion had beentreated with a laxative, poloxalene, and letting the air out with ahose. However, the animal bloated again, and on the second occasion'wastreated with a two-gram gelatin capsule of our bloat enzyme (2000units). The relieved the bloat immediately. He was observed for threedays thereafter, and during this time had no further recurrence ofbloat.

' In the aforesaid examples only the weight of the enzyme in the capsuleis stated. Lactose was added in each instance to take up any remainingair space in the capsule.

What is claimed is:

l. The method of preparing an enzyme product effective for the controlof bloat that comprises culturing Streptomyces griseus NRRL 5747 in afermentation medium until said enzyme has the following characteristics:

' a.-1 g. of enzyme represents about 500 units, 1 unit being that amountof enzyme which when dissolved in 1 ml. of water reduces the viscosityof 1 ml. of prepared gastric mucin by 50% in 2 hours at 39 C.;

b. said enzyme is insoluble in acidic acetone;

c. centrifuged broth from the fermentation medium contains 0.3 1.0 unitsof enzyme/mg. broth;

(1. storage of the dry enzyme at F. for 28 weeks or at 122 F. for8 weeksresults in less than 10% activity decrease;

and recovering from the fermentation liquor an enzyme product effectivefor the control of bloat.

2. The method according to claim 1 in which the recovery includescentrifugation of the fermentation liquor, acidifying the recoveredliquor, extracting it with acetone, and recovering the enzyme product byremoval of acetone.

3. The enzyme product resulting from the method of claim 1. v

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. I 3 898DATED I August 5, 1975 |NVENTOR(5) 3 Peter A. Hahn, Frank J. Hartdegen,Marlin A. Espen- .shade t It rs certrfred that error appearsm theabove-rdenhhed patent and that said Letters Patent are hereby correctedas shown below:

In Column 8, Claim 1, line 41:

"d. storage of the dry enzyme at 190 F. for 28 weeks should read:

"d. storage of the dry enzyme at 109 F. for 28 weeks" Signed and Sealedthis [SEAL] Arrest.-

liwntc. Meson c. MARSHALL DANN HeslmgOj/wer (ummissr'om-r /Pau'nls andTrademarks thirtieth Day of Septemberl975

1. THE METHOD OF PREPARING AN ENZYMES PRODUCT EFFECTIVE FOR THE CONTROLOF BLOAT THAT COMPRISES CULTURING STREPTOMYCES GRISEUS NRRL 5747 IN AFERMENTATION MEDIUM UNTIL SAID ENZYMES HAS THE FOLLOWINGCHARACTERISTICS: A. 1 G. OF ENZYMES REPRESENTS ABOUT 500 UNITS 1 UNITBEING THAT AMOUNT OF ENZYMES WHICH WHEN DISSOLVED IN 1 ML. OF WATERREDUCES THE VISCOSITY OF 1 ML OF PREPARED FASTRIC. MUCIN BY 50% IN 2HOURS AT 39*C, B. SAID ENZYMES IS INSOLUBLE IN ACIDIC ACETONE, C.CENTRIFUGED BROTH FROM THE FERMENTATION MEDIUM CONTAINS 0.3-1.0 UNITS OFENZYMES/MG BROTH D. STORAGE OF THE DRY ENZYME AT 190*F FOR 28 WEEKS ORAT 122*F FOR 8 WEEKS RESULTS IN LESS THAN 10% ACTIVITY DECREASE, ANDRECOVERING FROM THE FERMENTATION LIQUOR AN ENZYMES PRODUCT EFFECTIVE FORTHE CONTROL OF BLOAT.
 2. The method according to claim 1 in which therecovery includes centrifugation of the fermentation liquor, acidifyingthe recovered liquor, extracting it with acetone, and recovering theenzyme product by removal of acetone.
 3. The enzyme product resultingfrom the method of claim 1.